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Biology of Reproduction 60, 405-412 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Circulating Concentrations of Estradiol, Luteinizing Hormone, and Follicle-Stimulating Hormone during Waves of Ovarian Follicular Development in Prepubertal Cattle1

E.J. Melvin3,a, B.R. Lindseya, J. Quintal-Franco4,a, E. Zanellaa, K.E. Fikea, C.P. Van Tassell5,b, and J.E. Kinder2,a

a Department of Animal Science, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0908 b Roman L. Hruska Meat Animal Research Center, USDA, ARS, Clay Center, Nebraska 68933-0166


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Objectives were to characterize changes in concentrations of LH, FSH, and estradiol-17ß (estradiol) in blood and populations of ovarian follicles of prepubertal cattle during waves of follicular development and to evaluate interactions between day after follicular aspiration and month prepuberty for these variables. During each month (month prepuberty), ovarian follicles of prepubertal cattle were aspirated to induce synchronous emergence of a wave of follicular development (day after follicular aspiration). Characteristics of ovarian follicular development and concentrations of hormones in blood were evaluated during the synchronous wave of follicular growth. Multiple regression was used to estimate hormonal variables and evaluate interactions for variables between day after follicular aspiration and month prepuberty. There were no interactions between day after follicular aspiration and month prepuberty for numbers of follicles <= 4 or > 5 mm or concentrations of LH, FSH, and estradiol. The pattern of change in these variables after follicular aspiration was, therefore, similar each month prepuberty. There were interactions between day after follicular aspiration and month prepuberty for frequency and amplitude of LH pulses and size of largest follicle. There were also endocrine changes that were related to follicular development after follicular aspiration throughout the peripubertal period.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovariectomy of prepubertal cattle results in an increase in frequency of release of LH pulses [1–3]. This post-ovariectomy increase in frequency of LH pulses can be inhibited by administration of estradiol [3, 4]. The number of receptors for estradiol in the medial basal hypothalamus decreases prepuberty, and this decrease may result in the decreased negative feedback of estradiol on release of LH pulses [5]. Thus there is thought to be a change in sensitivity to estradiol due to decreased receptors at the hypothalamus in prepubertal cattle, with estradiol produced by ovarian follicles regulating secretion of LH by inhibiting the putative hypothalamic pulse generator for LHRH. Consequently, frequency of release of LH pulses increases between 50 days prepuberty and onset of puberty [5].

Prepubertal cattle have waves of ovarian follicular development similar to those observed in postpubertal cattle [6–8]. Associated with these waves of ovarian follicular development are changes in diameter of dominant follicles and of largest subordinate follicles [8]. As with postpubertal female cattle, increases in concentrations of FSH in blood plasma have been reported to precede emergence of waves of follicular development in prepubertal cattle [9].

Development of the largest ovarian follicle of postpubertal cattle can be classified into selection, growth, and plateau phases, and all follicles that do not ovulate subsequently go through an atretic phase [10]. Circulating concentrations of estradiol are elevated during the growth phase of dominant follicles when compared with circulating concentrations during plateau and atretic phases. Associated with this increase in circulating concentrations of estradiol is a greater frequency of release of LH pulses. Circulating concentrations of estradiol are lower when dominant follicles attain maximum diameters than during the growth phase of the development of dominant follicles. As concentrations of progesterone increase with corpus luteum development in cattle, estradiol in blood serum decreases, probably as a result of the decreased frequency of LH pulses between the growth and plateau phases of the wave of follicular development [11]. The amount of aromatase mRNA is small until an ovarian follicle is between 4 and 5 mm in diameter in adult cattle [12]. Removal of follicles >= 4 mm from the ovaries of prepubertal cattle would therefore allow for evaluation of the role of estradiol and other factors contained in follicles in regulation of LH and FSH release during waves of follicular development. In the present experiment, we used the technique of aspiration of ovarian follicles >= 4 mm to study endocrine changes and antral follicular replenishment during waves of follicular development at monthly intervals preceding puberty in female cattle.

We hypothesized that >= 4-mm ovarian follicles of prepubertal cattle released factors in the blood, particularly estradiol, in varying amounts during waves of ovarian follicular development, which regulated the release of LH and FSH in a dynamic fashion as the wave of follicular development progressed. More importantly, we hypothesized that there would be changes in release of these ovarian factors and changes in how these factors regulated release of LH and FSH in female cattle during waves of ovarian follicular growth as processes of sexual maturation developed in the months preceding puberty.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian Follicular Development

Nine prepubertal female cattle (Angus, n = 5; Saler, n = 2; Braler, n = 2; 9 mo of age at the initiation of the experiment; 258 ± 25 kg) were used in the study. Age at puberty for each female was determined retrospectively on the basis of the time when luteal function was initially detected (concentrations of progesterone > 1 ng/ml of blood plasma). To synchronize the stages of ovarian follicular development among females (day after follicular aspiration), ovarian follicles >= 4 mm in diameter were aspirated via the transvaginal approach [13] at 9 mo of age, a second time at 11 mo of age, and monthly thereafter at approximately the same time each month until puberty (day prepuberty). Ovarian follicular development was evaluated by ultrasonography on the day subsequent to follicular aspiration (Day 1) and every day thereafter for 9 days (Day 0 = day of follicular aspiration). Ovarian follicles were classified as < 5 mm, >= 5 mm, or largest follicles depending on diameter. The largest follicle in each case was retrospectively identified as the follicle with the greatest diameter after follicular aspiration.

Concentrations of Hormones

To determine age at puberty, blood samples were collected twice weekly beginning at 7 mo of age and continuing until puberty as defined by onset of luteal function, which was determined by circulating concentrations of progesterone. Concentrations of serum progesterone > 1 ng/ml from blood samples collected twice weekly for two consecutive samples were used as the criteria to determine time of puberty.

To collect serial blood samples after follicular aspiration, all females were acclimated to human contact and stanchions 1 mo before initiation of serial blood collections. Experimental animals were fitted with an indwelling jugular catheter 6 h before aspiration of ovarian follicles. Serial blood samples were collected at 6-h intervals starting at the time of ovarian follicular aspiration (Day 0), and sampling continued through Day 9 after follicular aspiration to quantify circulating concentrations of FSH and estradiol. Serial blood samples were collected daily starting at time of ovarian follicular aspiration (Day 0), and sampling continued through Day 9 after follicular aspiration to quantify circulating concentrations of progesterone. More frequent serial blood samples to characterize the secretory pattern of LH (frequency and amplitude of LH pulses) were collected at 20-min intervals for 12 h immediately after ovarian follicular aspiration (Day 0). The frequent serial blood collections over the 12-h period to evaluate the pattern of LH secretion were repeated at Days 2, 4, 6, and 8 after follicular aspiration.

The initial 9-day serial blood collections and ovarian follicle evaluations were conducted when females were 9 mo of age. The experimental protocol of ovarian follicular aspiration, ultrasonography of ovarian structures, and blood sampling was repeated at 11 mo of age and subsequently at monthly intervals until puberty in each female.

Hormone Quantitation and Statistical Analysis

Concentrations of LH were determined by RIA [14], and these data were analyzed by Pulsar (Pulsar software modified for IBM-PC by J.F. Gitzen and V.D. Ramirez, Urbana, IL) to evaluate frequency of release and amplitude of LH pulses, and mean and basal concentrations of serum LH. "G" values used with the Pulsar program to evaluate variables of LH secretion were 5.5, 4.4, 1.6, 1.3, and 10.0 for G (1) to G (5), respectively. Intra- and interassay coefficients of variation for standard sera were 8.8 and 10.4%, and 11.0 and 12.1%, respectively. Circulating concentrations of FSH were quantified by RIA [14]. Intra- and interassay coefficients of variation for standard sera were 7.7 and 8.6% and 10.6 and 13.1%, respectively. Circulating concentrations of estradiol were quantified by RIA [15]. Intra- and interassay coefficients of variation for standard sera were 8.4 and 22.8%, respectively.

Circulating concentrations of progesterone were quantified by RIA. Concentrations of progesterone (nonextracted) in plasma were assayed by using a Coat-A-Count assay kit (Diagnostic Product Corporation, Los Angeles, CA). The standard curve was modified by the addition of an extra point to the curve at 5 ng/ml, and three additional quality-control samples were also included using plasma with low, medium, and high concentrations of progesterone. The assay was validated by pipetting sample volumes of 100, 50, 25, and 12.5 µl and bringing the volume to 100 µl by adding Calibrator A (provided in the kit). Each sample volume was used with samples containing low, medium, or high concentrations of progesterone. Parallelism was determined by using the Allfit program [16]. Slopes of dilutions of plasma and the standard curve were not different as determined by the Allfit program. Recovery of added mass averaged 89% for three different amounts of progesterone (1, 5, and 10 ng/ml) added to 50 µl from bovine plasma of three different pools containing either high, medium, or low concentrations of progesterone.

Data were analyzed using polynomial multiple regression analyses including the effects of day after follicular aspiration and month prepuberty [17]. Effects of day after follicular aspiration and month prepuberty were included with polynomial regression effects tested up to the fourth power. Sequential sums of squares were used to eliminate regression variables from the highest to the lowest power. Interaction effects (between day after follicular aspiration and month prepuberty) with combined powers up to four were also tested. The final model included all the interactions up to the highest interaction that was significant and the main effects. An estimated multiple regression equation was generated from each analysis, and this equation was used to determine estimated values (endocrine and ovarian follicular variables) for the days after follicular aspiration and for each of the 8 mo prepuberty. Because time to puberty for each female was not known at initiation of the experiment and age at puberty was determined retrospectively, data for different numbers of females were included in the analyses at each month prepuberty (8 mo, n = 6; 7 mo, n = 4; 6 mo, n = 7; 5 mo, n = 8; 4 mo, n = 8; 3 mo, n = 9; 2 mo, n = 9; 1 mo, n = 7). Two females had their pubertal ovulation during the final 9-day bleeding period for these females. Data from these two females were not included in the 1-mo statistical analyses; consequently there were data for only 7 females at 1 mo prepuberty.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Numbers of Follicles < 5 mm in Diameter

There was no interaction between day after follicular aspiration and month prepuberty for number of follicles < 5 mm, indicating that there was no change in the pattern of development in the number of follicles < 5 mm after follicular aspiration over the 8-mo prepubertal period. During the 8 mo prepuberty, the number of follicles < 5 mm increased (quadratic regression, p < 0.0001; Fig. 1a) between 8 and 5 mo prepuberty, with a greater increase in the number of follicles < 5 mm between 8 and 7 mo prepuberty than between 7 and 6, and 6 and 5 mo prepuberty. The number of follicles < 5 mm decreased between 5 and 1 mo prepuberty, with a greater magnitude of decline in the number of follicles between 2 and 1 mo prepuberty than between 5 and 4, 4 and 3, and 3 and 2 mo prepuberty. The number of follicles < 5 mm increased (linear regression, p < 0.0001; Fig. 1b) between Day 1 and Day 9 after follicular aspiration.



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  		FIG. 1. Estimated mean number of follicles < 5 mm in diameter for the 9 days after aspiration of all follicles >= 4 mm in diameter (day after follicular aspiration) each month from 8 to 1 mo prepuberty (month prepuberty). There was no interaction between day after follicular aspiration and month prepuberty; therefore, data for the two variables are depicted separately; a) a quadratic regression coefficient was used to estimate values for month prepuberty (p < 0.0001), and b) a linear regression coefficient was used to measure values for day after follicular aspiration (p < 0.0001). SEM = 0.41.

Numbers of Follicles >= 5 mm in Diameter

There was no interaction between day after follicular aspiration and month prepuberty for the number of follicles >= 5 mm, indicating that there was no change in the pattern of development in the number of follicles >= 5 mm after follicular aspiration over the 8-mo prepubertal period. The number of follicles >= 5 mm increased (quadratic regression, p < 0.02; Fig. 2a) between 8 and 3 mo prepuberty and remained constant thereafter until puberty. The number of follicles >= 5 mm increased (cubic regression, p < 0.0001; Fig. 2b) between Days 1 and 4 after follicular aspiration, with the number of follicles on Day 1 the least during the nine days after follicular aspiration. There was a large increase in the number of follicles >= 5 mm between Days 1 and 2, and a more gradual increase in the number of follicles >= 5 mm between Days 2 and 4 after follicular aspiration. The number of follicles >= 5 mm decreased gradually between Days 4 and 7 after follicular aspiration. There was a second increase in the number of follicles >= 5 mm between Days 7 and 9 after follicular aspiration.



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  		FIG. 2. Estimated mean number of follicles >= 5 mm in diameter for the 9 days after aspiration of all follicles >= 4 mm in diameter (day after follicular aspiration) each month from 8 to 1 mo prepuberty (month prepuberty). There was no day after follicular aspiration by month prepuberty interaction; therefore, data for the two variables are depicted separately; a) a quadratic regression coefficient was used to estimate values for month prepuberty (p < 0.02), and b) a cubic regression coefficient was used to estimate values for day after follicular aspiration (p < 0.0001). SEM = 0.18.

Size of the Largest Ovarian Follicle

There was an interaction (p = 0.03) between day after follicular aspiration and month prepuberty for the size of the largest follicle, indicating a change in the pattern of development of the largest ovarian follicle after follicular aspiration during the 8-mo prepubertal period. The largest follicle in the ovaries grew an average of 6.8 mm between Days 1 and 9 after follicular aspiration each month prepuberty, and rate of growth of the largest follicle increased gradually (Fig. 3) as females approached puberty. There was an interaction (p = 0.03) between day after follicular aspiration and month prepuberty as a result of a more rapid rate of growth of the largest follicle after follicular aspiration at 1 than at 8 mo prepuberty. Diameter of the largest follicle increased (quadratic regression, p < 0.0001; Fig. 3) between Days 1 and 5, with a more gradual increase between Days 5 and 9 after follicular aspiration. After follicular aspiration, the diameter of the largest follicle increased between Days 1 and 5 (4.7 mm at 8 and 5.7 mm at 1 mo prepuberty), with a more gradual increase in diameter between Days 5 and 9 at 8 mo (1 mm) compared with a more rapid growth (2 mm) 1 mo prepuberty.



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  		FIG. 3. Estimated mean diameter of the largest follicle for the 9 days after aspiration of all follicles >= 4 mm in diameter (day after follicular aspiration) from 8 to 1 mo prepuberty (month prepuberty). There was a day after follicular aspiration by month prepuberty interaction; therefore, data are depicted three-dimensionally (p = 0.03); a quadratic regression coefficient was used to estimate values for day after follicular aspiration (p < 0.0001). SEM = 0.07.

Circulating Concentrations of FSH

There was no interaction between day after follicular aspiration and month prepuberty for concentrations of FSH, indicating that there was no difference in the pattern of change in circulating concentrations of FSH after follicular aspiration during the 8-mo prepubertal period. Concentrations of FSH increased (linear regression, p < 0.004; Fig. 4a) over the 8-mo prepubertal period. Concentrations of FSH decreased (quartic regression; p < 0.007; Fig. 4b) between Days 0 and 5, with the lowest values observed on Day 5 after follicular aspiration each month prepuberty. The decline in circulating concentrations of FSH was of greatest magnitude from Days 2 through 4 after follicular aspiration. Concentrations of FSH in plasma increased between Days 5 and 8 and decreased between Days 8 and 9 after follicular aspiration each month prepuberty.



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  		FIG. 4. Estimated mean circulating concentrations of FSH in blood plasma (ng/ml) for the 9 days after aspiration of all follicles >= 4 mm in diameter (day after follicular aspiration) each month from 8 to 1 mo prepuberty (month prepuberty). There was no day after follicular aspiration by month prepuberty interaction; therefore, data for the two variables are depicted separately; a) a linear regression coefficient was used to estimate values for month prepuberty (p < 0.004), and b) a quartic regression coefficient was used to estimate values for day after follicular aspiration (p < 0.007). SEM = 0.04.

Mean Concentrations of Serum LH

There was no interaction between day after follicular aspiration and month prepuberty for concentrations of LH, indicating that there was no difference in the pattern of change in circulating concentrations of LH after follicular aspiration during the 8-mo prepubertal period. Concentrations of LH decreased (quartic regression, p < 0.008; Fig. 5a) between 8 and 7 mo prepuberty and increased between 7 and 4 mo prepuberty. There was a subtle decline in the concentration of LH in the blood between 4 and 3 mo prepuberty. The concentration of LH in blood serum increased between 3 and 1 mo prepuberty, with the greatest concentration during the 8 mo of the prepubertal period observed at 1 mo prepuberty. The concentration of LH decreased (quadratic regression, p < 0.03; Fig. 5b) between the time of follicular aspiration and Day 8 after follicular aspiration each month prepuberty.



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  		FIG. 5. Estimated mean concentrations of LH in blood serum (ng/ml) for the 8 days after aspiration of all follicles >= 4 mm in diameter (day after follicular aspiration) each month from 8 to 1 mo prepuberty (month prepuberty). There was no day after follicular aspiration by month prepuberty interaction; therefore, data for the two variables are depicted separately; a) a cubic regression coefficient was used to estimate values for month prepuberty (p < 0.008), and b) a quadratic regression coefficient was used to estimate values for day after follicular aspiration (p < 0.03). SEM = 0.15.

Amplitude of LH Pulses

There was an interaction (linear regression; p < 0.03) between day after follicular aspiration and month prepuberty for amplitude of LH pulses, indicating a change in the pattern of amplitude of LH pulses after follicular aspiration during the 8-mo prepubertal period. At 8 mo prepuberty, there was a subtle increase in the amplitude of LH pulses over the 8 days after follicular aspiration, but a dramatic decline from 3 to 1 mo prepuberty in the amplitude of LH pulses (Fig. 6) between Days 0 and 6 after follicular aspiration.



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  		FIG. 6. Estimated mean amplitude of LH pulses (ng/ml blood serum) for the 8 days after aspiration of all follicles >= 4 mm in diameter (day after follicular aspiration) each month from 8 to 1 mo prepuberty (month prepuberty). There was a day after follicular aspiration by month prepuberty interaction; therefore, data are depicted three-dimensionally (p < 0.03); a linear regression coefficient was used to estimate values for month prepuberty (p < 0.06) and values for day after follicular aspiration (p < 0.003). SEM = 0.09.

Frequency of Release of LH Pulses

There was an interaction between day after follicular aspiration and month prepuberty for the number of LH pulses, indicating a change in the pattern of frequency of LH pulses after follicular aspiration from 8 to 1 mo prepuberty. The number of LH pulses increased (linear regression, p < 0.05; Fig. 7) between 8 and 6 mo prepuberty throughout the follicular wave. The numbers of LH pulses were similar between 6 and 3 mo prepuberty. The number of LH pulses increased between 3 and 1 mo prepuberty on each day after follicular aspiration. At 8 mo prepuberty, the number of LH pulses decreased (cubic regression, p < 0.002; Fig. 7) between Days 0 and 6 after follicular aspiration, with the decline of greatest magnitude between Days 2 and 4 after follicular aspiration. The number of LH pulses increased between Days 6 and 8 after follicular aspiration. There was a similar pattern in change of frequency of LH pulses after follicular aspiration 7 and 6 mo prepuberty. At 5 and 4 mo prepuberty, the number of LH pulses increased between Days 0 and 2, decreased between Days 2 and 6, and increased between Days 6 and 8 after follicular aspiration. At 3, 2, and 1 mo prepuberty, the number of LH pulses increased between Days 0 and 2 and decreased between Days 2 and 8 after follicular aspiration.



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  		FIG. 7. Estimated mean number of LH pulses/12 h in blood serum for the 8 days after aspiration of all follicles >= 4 mm in diameter (day after follicular aspiration) each month from 8 to 1 mo prepuberty (month prepuberty). There was a day after follicular aspiration by day by month prepuberty interaction; therefore, data are depicted three-dimensionally; a linear regression coefficient was used to estimate values for month prepuberty (p < 0.05), and a cubic regression coefficient was used to estimate values for day after follicular aspiration (p < 0.002). SEM = 0.06.

Circulating Concentrations of Estradiol

There was no interaction between day after follicular aspiration and month prepuberty for concentrations of estradiol, indicating that there was no change in the pattern of circulating estradiol after follicular aspiration during the 8-mo prepubertal period. Over the 8-mo prepubertal period, changes in the concentration of estradiol were subtle, with concentrations tending to decrease from 8 through 3 mo prepuberty and increase (quartic regression, p < 0.03; Fig. 8a) from 3 to 1 mo prepuberty. The concentration of estradiol decreased (cubic regression, p < 0.008; Fig. 8b) between Days 0 and 1 each month prepuberty and increased between Days 1 and 7 after follicular aspiration each month prepuberty. The greatest magnitude of increase in the concentration of estradiol was between Days 2 and 6 after follicular aspiration each month prepuberty. The concentration of estradiol decreased between Days 7 and 9, with the average decrease between Days 7 and 8 after follicular aspiration being of lesser magnitude than the decrease between Days 8 and 9 after follicular aspiration during each month of the prepubertal period.



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  		FIG. 8. Estimated mean circulating concentrations of estradiol-17ß in blood plasma (pg/ml) for the 9 days after aspiration of all follicles >= 4 mm in diameter (day after follicular aspiration) each month from 8 to 1 mo prepuberty (month prepuberty). There was no day after follicular aspiration by month prepuberty interaction; therefore, data for the two variables are depicted separately; a) a quartic regression coefficient was used to estimate values for month prepuberty (p < 0.03) and b) a cubic regression coefficient was used to estimate values for day after follicular aspiration (p < 0.008). SEM = 0.18.

Circulating Concentrations of Progesterone

No samples had circulating concentrations of progesterone of > 1.0 ng/ml of plasma over the 9 days after aspiration of all ovarian follicles >= 4 mm in diameter, indicating that aspiration of ovarian follicles did not induce formation of functional luteal tissue.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Data from the present experiment indicate that the rate of growth of the largest follicle, and the number and amplitude of LH pulses change during waves of ovarian follicular development as the peripubertal period progresses. The rate of growth of the largest ovarian follicle was greater during the selection phase of follicular development, when the number of LH pulses was greater and concentrations of estradiol were lower, than during the growth phase of follicular development. As the largest follicle grew and developed during the early growth phase of follicular development peripuberty, concentrations of estradiol increased and the number of LH pulses decreased, resulting in a decreased rate of growth of the largest follicle. During the latter portion of the growth phase, the diameter of the largest follicle increased slightly, concentrations of estradiol decreased, and the number of LH pulses increased.

There was no interaction between day after follicular aspiration and month prepuberty for numbers of follicles < 5 or >= 5 mm or for concentrations of LH, FSH, or estradiol. This indicates that the pattern of change in these variables over the 9 days subsequent to follicular aspiration was similar each month during the 8-mo prepubertal period. Most important, however, are the changes in these variables during waves of ovarian follicular development in prepubertal cattle. The stage of the wave of ovarian follicular development did have a significant influence on secretion of LH, FSH, and estradiol. This indicates the intricate communication between the ovarian follicle and the hypothalamic-pituitary axis. Because the extent and type of communication to the hypothalamic-pituitary axis, via estradiol and other ovarian factors, change with stage of the wave of ovarian follicular development, evaluation of endocrine and physiological variables related to reproduction without consideration of the stage of the wave of ovarian follicular development limits the value of data obtained.

Of all the interactions evaluated between day after follicular aspiration and month prepuberty, the only interactions observed were for frequency and amplitude of LH pulses and size of the largest follicle. This interaction indicated there were changes in the pattern of release of LH pulses that enhanced development of the largest follicle after follicular aspiration as time progressed from 8 to 1 mo prepuberty. Previous research from our laboratory indicates that the increase in frequency of LH pulses during the peripubertal period is a primary regulator of onset of puberty [18]. The dramatic changes in the pattern of LH pulses (both frequency and amplitude) after follicular aspiration over 8 mo prepuberty serve to indicate the importance of change in the pattern of release of LH pulses in processes of sexual maturation of female cattle. This also further emphasizes the role of the secretory pattern of LH in stimulating development of the largest ovarian follicle in cattle.

The concentration of FSH was elevated after aspiration of ovarian follicles and decreased between Days 0 and 5 after follicle aspiration each month prepuberty. This finding is in agreement with previous research in which unilateral ovariectomy of prepubertal cattle resulted in a transient increase in FSH, with concentrations peaking at 24 h after removal of the ovary [19]. With unilateral ovariectomy of prepubertal cattle, the response in gonadotropin secretion to removal of the ovary depends on whether the ovary that is removed contains the large follicles, particularly the dominant follicle, that are involved in regulation of secretion of the gonadotropins. In the present study, the concentration of FSH increased between Days 5 and 8 after follicular aspiration, with this increase preceding the second increase in the number of follicles >= 5 mm. These data indicate that concentrations of FSH gradually increase 2–3 days before emergence of waves of ovarian follicular development during the 8-mo prepubertal period. The peak concentration of FSH during the period of increased FSH 5–8 days after follicular aspiration was lower than the peak concentration immediately following follicular aspiration. The most likely explanation for the differences in peak concentrations of FSH was the effect of follicular aspiration on synchronizing ovarian follicular development among animals.

A pattern of ovarian follicular development and change in the concentration of FSH in 8-mo-old prepubertal cattle similar to those detected in the present study has been previously reported, with the concentration of FSH elevated 1 day before the increase in the number of subordinate follicles (>= 4 mm) [7]. In postpubertal cattle, the concentration of FSH starts to increase 2–4 days before the emergence of waves of ovarian follicular development, and peaks 1 or 2 days before the emergence of those waves [11, 20]. The number of follicles >= 5 mm and the concentration of FSH increased between 8 and 3 mo prepuberty. Because increases in concentration of FSH precede the increase in number of follicles at initiation of a new wave of ovarian follicular development in prepubertal [7] and postpubertal [11, 20] cattle, it is possible that greater concentrations of FSH as puberty approached may have induced development of greater numbers of follicles >= 5 mm.

The selection phase of the largest follicle was between Days 1 and 4 after follicle aspiration, with the number of follicles >= 5 mm increasing rapidly during this time period. More rapid growth occurred, however, after aspiration of the largest follicle at 1 than at 8 mo prepuberty. Although the regression phase of the largest follicle in the present study was not apparent in terms of a decline in diameter of the largest follicle, the increase in follicles >= 5 mm between Days 7 or 8 and 9 after follicle aspiration indicates that the large ovarian follicle was beginning to lose dominance and a new wave of ovarian follicular development was being initiated. The number of subordinate follicles increases around the time of initiation of atresia of dominant follicles in 8-mo-old prepubertal [7] and postpubertal cattle [19, 21].

A possible explanation of the continuous increase in the number of follicles between Days 0 and 9 after follicular aspiration was the effect that aspiration had on the number of follicles < 5 mm. Removal of follicles >= 4 mm in diameter resulted in a lesser number of follicles < 5 mm on Day 1 after follicular aspiration as a result of ablating some follicles < 5 mm and removing larger follicles, which would have eventually decreased in diameter and entered the group of follicles < 5 mm in diameter as they underwent atresia.

The number of follicles < 5 mm increased between 8 and 5 mo prepuberty and decreased between 5 and 1 mo prepuberty. The most likely explanation for the increase in the number of follicles < 5 mm between 8 and 5 mo prepuberty was the increase in the concentration of FSH as females progressed from 8 to 5 mo prepuberty. Because an increase in the concentration of FSH precedes the emergence of waves of ovarian follicular development and is necessary for the initiation of new waves [20], it is possible that the increase in the concentration of FSH between 8 and 1 mo prepuberty resulted in an increase in the recruitment of follicles < 5 mm and more rapid development of follicles < 5 mm to follicles >= 5 mm. The increase in follicles >= 5 mm as puberty approached may have occurred because a larger number of follicles < 5 mm developed more rapidly to >= 5 mm because of the greater concentration of FSH, thus decreasing the number of follicles < 5 mm.

Changes in concentrations of estradiol were subtle and must be interpreted with caution. In prepubertal female cattle, however, the hypothalamus is exquisitely sensitive to estradiol feedback inhibition of LHRH-induced LH secretion, so differences, even though subtle, may have important physiological implications. As hypothesized, the concentration of estradiol decreased over the 24 h after follicular aspiration, indicating that removal of all follicles >= 4 mm in diameter decreased ovarian estrogen synthesis. However, this post-aspiration decrease in the concentration of estradiol was not acute but gradual over the 24 h after follicular aspiration. It is possible that aspiration of ovarian follicles did not immediately disrupt the ability of the granulosal cells to synthesize and release estradiol. Granulosal cells that remained in aspirated follicles may have undergone a gradual loss of steroidogenesis, which could explain why there was a gradual decrease in the concentration of estradiol over the 24 h after follicular aspiration. The decrease in the concentration of estradiol after removal of all follicles >= 4 mm in diameter and the subsequent increase in the concentration of estradiol during the growth period for follicles >= 5 mm support previously reported data [12] that follicles begin to have larger quantities of mRNA for aromatase when sizes of 4–5 mm in diameter are achieved.

The concentration of estradiol continued to increase between Days 5 and 7 after follicular aspiration, and this corresponded to the early portion of the growth phase of the largest follicle. The concentration of estradiol increased during the selection phase of the largest ovarian follicle that developed during the first wave of ovarian follicular growth after ovulation, and peak concentrations occurred during the early portions of the growth phase of dominant follicles in postpubertal cattle [22]. After this peak in the concentration of estradiol in blood plasma during the early portions of the growth phase, the concentration progressively decreased even though the largest follicle continued to grow in diameter for another four days. Similar data were previously reported for postpubertal cattle, with concentrations of estradiol in circulation being greater during the growth phase of first waves of ovarian follicular development of the estrous cycle than during the plateau phase of development, when the largest ovarian follicle attains maximum diameter [11]. Data from the present study indicate that the pattern of synthesis and release of estradiol by the granulosal cells of the largest follicle during waves of ovarian follicular growth is established as early as 8 mo prepuberty.

The diameter of the largest follicle increased as puberty approached, probably as a result of increased numbers of LH pulses. The number of LH pulses increased between Days 0 and 2 after follicular aspiration at 5, 4, 3, 2, and 1 mo prepuberty, and this increase could have resulted in the increased rate of growth of the largest follicle compared with that at 8, 7, and 6 mo prepuberty, when the number of LH pulses did not increase between Days 0 and 2 after follicular aspiration. The number of LH pulses increased between Days 6 and 8 after follicular aspiration at 8, 7, 6, 5, and 4 mo prepuberty, but the increase in diameter of the largest follicle was of lesser magnitude than that of the largest follicle at 3, 2, and 1 mo prepuberty. The increase in the number of follicles >= 5 mm 9 days after follicular aspiration indicates that a new wave of ovarian follicular growth was beginning and that the largest follicle was in the early portion of the atresia phase and therefore unresponsive to the increase in the number of LH pulses.

The number of LH pulses in postpubertal cattle was greater during the growth phase of the first waves of ovarian follicular development of the estrous cycle than during plateau and regression phases of development [11]. In the present study, the number of LH pulses was greater during the selection phase of development of the largest follicle than during the growth phase each month prepuberty. It is possible that the varying concentrations of estradiol during the selection, growth, and atresia phases of the largest follicle may have an effect in regulating the number of LH pulses in prepubertal female cattle that is similar to the effect of progesterone in postpubertal female cattle, because estradiol is the primary regulator of the number of LH pulses during the prepubertal period in female cattle [3, 14]. There was a prepubertal decline in negative feedback of estradiol on secretion of LH in ovariectomized prepubertal cattle administered estradiol implants during the period when age-matched intact female cattle attained puberty. After this period, the ovariectomized females treated with estradiol had greater amounts of LH in circulation because of an enhanced amplitude of LH pulses [5]. These data support the hypothesis that estradiol is the primary regulator of the number of LH pulses during waves of ovarian follicular development, with the inhibitory effect of estradiol on the frequency of LH pulses decreasing as puberty approaches.

In conclusion, concentrations of estradiol, FSH, and LH, and the number of LH pulses changed during the wave of ovarian follicular development as puberty approached. This supported our hypothesis that ovarian follicles of prepubertal female cattle release factors in the blood, particularly estradiol, in varying amounts during waves of ovarian follicular development that regulate release of LH and FSH in a dynamic fashion as the wave of follicular development progresses. During the wave of ovarian follicular development, the concentration of FSH exhibited changes similar to the pattern observed during waves of ovarian follicular growth in postpubertal females. The concentration of FSH increased as puberty approached, with this increase corresponding to an increase in the number of follicles >= 5 mm. The number of LH pulses was greater during the selection phase of development of the largest follicle, when concentrations of estradiol were lower, than during the early and latest portion of the growth phase, when concentrations of estradiol were higher. As puberty approached, the number of LH pulses and the concentration of estradiol increased during the wave of ovarian follicular development, indicating decreased negative feedback of estradiol on secretion of LH. This increase in the number of LH pulses corresponded to an increased growth rate of the largest follicle, which ultimately attained a size of approximately 13 mm in the month preceding ovulation. In a previous study with cattle, when the largest follicle attained a size of 13 mm, the pubertal ovulation ensued [23]. The most important finding in the present experiment supports our hypothesis that the release of ovarian factors such as estradiol and the regulation by these factors of release of LH and FSH during waves of ovarian follicular development change with the transition from prepuberty to puberty. On the basis of the present and previous research, we hypothesize that the increased release of LH pulses during waves of ovarian follicular development during the last 3 mo prepuberty results in development of ovarian follicles of greater diameter, which ultimately reach sizes of about 13 mm and produce enough estradiol to induce the preovulatory surge of gonadotropins and the behavioral estrus at puberty.


   ACKNOWLEDGMENTS
 
We are particularly appreciative of the expert statistical advice of Dr. Vicente Vega-Murillo and the conscientious help of Dr. Hector Jimenez-Severiano in developing the figures included in this manuscript. We thank Dr. Jerry Reeves for LH antisera; Dr. Leo Reichert, Jr. for purified LH and FSH; Dr. Jim Dias for FSH antisera; Dr. Norman Mason for 17ß-estradiol antisera; Dr. Ed Grotjan, Jr. for use of the Four Fit program for analyzing estradiol data; Candy Toombs, Freddie Kojima, and Machele Miller for their superb technical assistance with the RIAs; Karl Moline, Jeff Bergman, and Bob Browelit for their assistance with caring for the cattle; and Ellen Bergfeld, Chris Hagedorn, Jess Landin, Shawn Peters, Casey Ramsel, Michael Wehrman, and Carl Clymer for assistance in sample collection.


   FOOTNOTES
 
1 This work was supported by appropriated funds from the State of Nebraska and USDA CRGO NRI 93–37203–9068. Published as paper 11914, Nebraska Agricultural Research Division. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. Back

2 Correspondence: James E. Kinder, A224j Animal Sciences, University of Nebraska, Lincoln, NE 68583–0908. FAX: 402 472 6362; ansc502{at}unlvm.unl.edu Back

3 Current address: Bowman Gray School of Medicine, Wake Forest University, Winston Salem, NC 27106. Back

4 Current address: INIFAP-SAGAR, C.E. Moccocha., Apdo. Postal 100 Suc., Merida, Yucatan, Mexico. Back

5 Current address: Building 263, Room 102A, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705–2350. Back

Accepted: September 22, 1998.

Received: June 16, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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